In vitro methods for determining in vivo thrombotic events

ABSTRACT

Diagnostic systems, methods, polypeptides and antibodies for detecting the presence of C-terminal hGPIIb fragment of the platelet receptor GPIIb-IIIa in a body fluid sample are disclosed.

TECHNICAL FIELD

[0001] The present invention relates to a fragment of the C-terminalregion of GPIIb heavy chain (hGPIIb) that is produced in vivo byindigenous protease cleavage of the GPIIb-IIIa platelet glycoproteinreceptor, and to in vitro detection of the fragment in body fluids ofindividuals undergoing thrombotic events. The present invention alsorelates to polypeptide analogs of the hGPIIb C-terminal fragment, andantibodies that immunoreact with the C-terminal fragment and todiagnostic methods for detecting thrombi and clotting disorders.

BACKGROUND

[0002] Thrombosis involves cell adhesion of platelets. Platelet celladhesion generally involves recognition of specific adhesive proteins bythe platelet cell surface receptor GPIIb-IIIa.

[0003] GPIIb-IIIa is a noncovalent, Ca⁺⁺ dependent, heterodimer complexcomprised of noncovalently associated alpha and beta subunits. Jenningset al., J. Biol. Chem., 257:10458-10466 (1982). The alpha subunit, GPIIbconsists of a heavy chain (hGPIIb) having a relative molecular weight ofabout 120 kilodaltons (KDa), and a light chain (lGPIIb) of about 20 KDathat are linked together by disulfide bonds. The beta subunit, GPIIIa isa single chain polypeptide of about 100 KDa. Phillips et al., J. Biol.Chem., 252:2121-2126 (1977).

[0004] GPIIb-IIIa contributes to platelet function through interactionswith RGD-containing proteins such as fibrinogen [Bennett et al., Proc.Natl. Acad. Sci. USA, 80:2417-2421 (1983)], fibronectin [Ginsberg etal., J. Clin. Invest., 71:619-624 (1983)], and von Willebrand factor[Ruggeri et al., Proc. Natl. Acad. Sci. USA, 79:6038-6041 (1982)], andtherefore is a component of the common platelet adhesive proteinreceptor [Pytela et al., Science, 231:1559-1562 (1986) and Plow et al.,J. Biol. Chem., 259:5388-5391 (1984)].

[0005] The sites on GPIIb-IIIa that are required to function as anadhesion receptor are not well characterized. Several observationssuggest that a functionally significant site on GPIIb-IIIa is near theepitope defined by the monoclonal antibody PMI-1. This antibody binds tothe heavy chain of GPIIb [Shadle et al., J. Cell. Biol., 99:2056-2060(1984)] and defines a region of GPIIb that is associated with severaldistinct functional activities. First, PMI-1 inhibits adhesion of washedplatelets to collagen. Shadle et al., J. Cell. Biol., 99:2056-2060(1984). Second, the surface orientation of this region is regulated bydivalent cations because millimolar (mM) concentrations of calcium ormagnesium suppress expression of the PMI-1 epitope. Ginsberg et al., J.Clin. Invest., 78:1103-1111 (1986). Third, abnormal divalent cationregulation of the conformation of this site is associated with afunctional thrombasthenic state. Ginsberg et al., J. Clin. Invest.,78:1103-1111 (1986). Fourth, stimulation of platelets with up to 100micromolar adenosine diphosphate (ADP) or epinephrine, 1 unit permilliliter thrombin, or 50 micrograms per milliliter calf skin collagendoes not substantially increase the binding of PMI-1 antibodies toplatelets.

[0006] Platelet activation has been reported to produce the appearanceof antigenic sites on the platelet surface that are not present in thenon-activated platelet, and at least one of such induced sites has beenlocalized to the GPIIb-IIIa receptor complex. Shattil et al, J. Biol.Chem. 260:11107-11114 (1985); Coller, B.S., J. Cell Biol., 103:451-456(1986).

[0007] Numerous assays based on the release of fragments of bloodcoagulation enzymes or platelet activation have been developed inattempts to identify thrombotic and pre-thrombotic states. From theplatelet perspective, initial efforts were focused on release ofplatelet specific antigens. Ginsberg, et al., In Immunologic Analysis:Recent Progress in Diagnostic Labor. Immunol., Nakamura et al., Eds.Masson Publishing, USA Inc., 199-209 (1982). Recent studies have focusedon the use of monoclonal antibodies selective for activated platelets.Frelinger et al., J. Biol. Chem., 263:12397-12402 (1988); Aiken et al.,Sem. Thromb. Hemost., 13:307-316 (1987); Berman et al., J. Clin.Invest., 78:130-137 (1986); Hsu-Lin et al., J. Biol. Chem.,259:9121-9126 (1984); George et al., J. Clin. Invest., 78:340-348(1986); Shattil et al., Blood, 73:150-158 (1989); Shattil et al., J.Biol. Chem., 260:11107-11114 (1985); Adelman et al., Blood, 70:1362-1366(1987).

[0008] Each of the above methods has been plagued by unintentional invitro activation because it is difficult to obtain blood samples in amanner such that the platelet activation events are not triggered duringvenipuncture, sample handling or the like. This difficulty has severelylimited the clinical utility of such assays. It would be desirable,therefore, to develop a method to detect the occurrence of thromboticevents without relying on platelet activation events. The utility ofdetermining the elevation in a plasma glycocalicin, as a potential assayfor in vivo platelet activation, has also been reported. Steinberg etal., N. Engl. J. Med., 317:1037-1042 (1987); Coller et al., J. Clin.Invest., 73:794-799 (1984).

[0009] It has recently been found that a class of antigenic determinantsare expressed when GPIIb-IIIa specifically binds to its ligand. Theantigenic determinants are not expressed by either the non-occupiedGPIIb-IIIa receptor or the non-bound ligand. One such determinantlocated on the C-terminus of the GPIIB heavy chain is recognized by thepreviously described PMI-1 monoclonal antibody. Shadle et al., J. CellBiol., 99:2056-2060 (1984); Frelinger et al., J. Biol. Chem.,263:12397-12402 (1988).

BRIEF SUMMARY OF THE INVENTION

[0010] It has now been found that a portion of the C-terminus of hGPIIb,containing the PMI-1 antigenic determinant, becomes exposed uponreceptor-ligand binding and is cleaved by indigenous proteases inducedby thrombus formation to form a cleaved fragment. The fragment, referredto as C-terminal hGPIIb fragment, is released into the vascular fluid,and finds its way into other body fluids, such as urine.

[0011] Simple activation of platelets with agonists in vitro is notsufficient to release the C-terminal hGPIIb fragment. Treatment with aprotease is required. In the thrombus, the platelet receptor isoccupied, thereby exposing this region of the molecule, and a varietyindigenous proteases, such as plasmin, are generated. Thus, in thepresent invention, detecting the release of C-terminal hGPIIb fragmentby proteases is not subject to the problems of in vitro activation, andcan be performed on a routinely obtained plasma or urine specimen from apatient.

[0012] The principal utility of the present invention is its ability toidentify in vitro thrombotic events ongoing in vivo, and to monitorthrombolysis. Clinical settings include acute thrombotic events such asstroke or coronary thrombosis, or more chronic events such as deepvenous thrombosis. In addition, detection of chronic ongoing thromboticevents can serve to identify individuals at risk of developing acutethrombosis.

[0013] The present invention is directed to a substantially isolatedC-terminal hGPIIb fragment that has a molecular weight of about 3900daltons, comprising an amino acid residue sequence that corresponds tothe carboxy terminal portion of the amino acid residue sequence ofhGPIIb shown in SEQ ID NO 1, including the amino acid residue sequencerepresented by the formula

-IHPAHHK-,

[0014] shown in SEQ ID NO 1 from residue 194 to residue 200. Thisfragment has the capacity to immunoreact with the monoclonal antibodyPMI-1, and preferably has the amino acid residue sequence comprising theamino acid residue sequence shown in SEQ ID NO 1 from about residue 173to about residue 200.

[0015] Also contemplated is a hGPIIb analog comprising a polypeptide ofno more than about 200 residues, having an amino acid residue sequencethat corresponds to the sequence of hGPIIb shown in SEQ ID NO 1 andincludes at least about 7 contiguous amino acid residues from the aminoacid residue sequence of hGPIIb shown in SEQ ID NO 1 from residue 173 toresidue 200. A preferred analog is a polypeptide comprising an aminoacid residue sequence corresponding to the formula:

-PQPPVNPLK-; -PQPPVNLPLKVDWGLPIPSPSP-; -PLKVDWGLPIP-; -PIPSPSPIHPAHHK-;or-IHPAHHK-

[0016] having a sequence shown in SEQ ID NO 1 from residue 173 toresidue 181, from residue 173 to residue 193, from residue 179 toresidue 189, from residue 187 to residue 200, or from residue 194 toresidue 200.

[0017] Further contemplated is an antibody comprising an antibodymolecule or fragment thereof that is capable of immunoreactingspecifically with C-terminal hGPIIb fragment but does not substantiallyimmunoreact with a polypeptide represented by the formula REQNSLDSWGPKhaving the sequence in SEQ ID NO 1 from residue 113 to residue 124. Inpreferred embodiments, the antibody composition contains a monoclonalantibody or fragment thereof that is substantially similar to theantibody, or fragment of the antibody, produced by hybridoma PMI-1.

[0018] The present invention also contemplates a method for assaying thepresence of a C-terminal hGPIIb fragment in a body fluid sample. Themethod comprises admixing an aliquot of body fluid with an antibody ofthis invention. Contact of the body fluid and the antibody is maintainedfor a time period sufficient for an immunoreaction product to form. Thefragment-containing immunoreaction product formed is then detected,thereby indicating the presence of the C-terminal hGPIIb fragment in thesample.

[0019] A diagnostic system in kit form is further contemplated by thepresent invention. In one embodiment is a diagnostic system in kit formfor assaying for the presence of a C-terminal hGPIIb fragment in a bodyfluid sample. The system comprises a package containing, in an amountsufficient to perform at least one assay, an antibody of this invention.The antibody can be in a liquid solution but in one emobodiment ispreferably attached to a solid-phase matrix. The system may also containa label for indicating the presence of the antibody molecules in theimmunoreaction product formed.

[0020] In a more preferred embodiment, the diagnostic system furthercontains a solid support comprising a solid matrix having affixedthereto at least one of the set of hGPIIb analog and a polypeptidehaving an amino acid residue sequence that includes the sequence of aC-terminal hGPIIb fragment, thereby providing a kit for assaying by acompetition ELISA a vascular fluid or urine sample for the presence ofC-terminal hGPIIb fragment.

[0021] In one preferred embodiment, the before-described method isutilized to assess the extent of acute thrombosis by detecting thepresence of C-terminal hGPIIb fragment in a body fluid sample andrelating the amount of immunoreaction product formed to predeterminedstandards correlative with the diseased state.

[0022] In another preferred embodiment, this method is useful to monitorthe course of plasminogen activator-induced thrombolysis by detectingthe presence of C-terminal hGPIIb fragment in a body fluid sample andrelating the amount to correlative predetermined levels.

[0023] In yet another preferred embodiment, this method is contemplatedfor identifying individuals at risk of developing acute thrombosis,which method determines the extent of chronic thrombosis and relates itto predetermined levels correlating with the risk of acute thrombosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In the Figures forming a portion of the disclosure of thisinvention:

[0025]FIG. 1 illustrates the effect of in vitro incubation of plateletsat 37CBP in the presence of 30 uM chymotrypsin as described in Example1c. One portion of the digested platelet suspension was centrifuged andthe supernatant (open diamond) and resuspended pellet (+) were assayedseparately. An equal portion of the platelet suspension was assayedcomprising total PMI-1 immunoreactive material (open square). Levels ofthe PMI-1 epitope (FIG. 1A) or the Tab epitope (FIG. 1B) (a monoclonalantibody with a different GPIIb epitope specificity) were determined bycompetitive ELISA assays, as described hereinafter. Note the release ofmaterial reactive with PMI-1, but not with Tab, into the platelet-freesupernatant.

DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Definitions

[0027] Amino Acid: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues identified herein are preferably in the natural “L”isomeric-configuration. However, residues in the “D” isomeric form canbe substituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxy terminus of thepolypeptide. In keeping with standard polypeptide nomenclature, J. Biol.Chem., 243:3557-59 (1969) and adopted at 37 C.F.R. 1.822(b)(2),abbreviations for amino acid residues are as shown in the followingTable of TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID YTyr L-tyrosine G Gly glycine F Phe L-phenylalanine M Met L-methionine AAla L-alanine S Ser L-serine I Ile L-isoleucine L Leu L-leucine T ThrL-threonine V Val L-valine P Pro L-proline K Lys L-lysine H HisL-histidine Q Gln L-glutamine E Glu L-glutamic acid W Trp L-tryptophan RArg L-arginine D Asp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

[0028] It should be noted that all amino acid residue sequences arerepresented herein by have a left to right orientation in theconventional direction of amino-terminus to carboxy-terminus. Inaddition, the phase “amino acid residue” is broadly defined to includemodified and unusual amino acids, such as those listed in 37 C.F.R.1.822(b)(4), and are incorporated by reference. Furthermore, it shouldbe noted that a dash at the beginning or end of an amino acid residuesequence indicates a peptide bond to a further sequence of one or moreamino acid residues or a covalent bond to an amino-terminal group suchas NH₂ or acetyl or to a carboxy-terminal group such as COOH.

[0029] Polypeptide and Peptide: Polypeptide and peptide are terms usedinterchangeably herein to designate a linear series of no more thanabout 50 amino acid residues connected one to the other by peptide bondsbetween the alpha-amino and carboxy groups of adjacent residues.

[0030] Protein: Protein is a term used herein to designate a linearseries of greater than 50 amino acid residues connected one to the otheras in a polypeptide.

[0031] II. Introduction

[0032] Platelets play a central role in thrombosis. The inventionprovides an in vitro means to detect platelet incorporation into thrombiin vivo, and thereby detects thrombotic events.

[0033] When platelets are incorporated into a thrombus, occupancy ofGPIIb-IIIa receptor by its ligand is believed to expose the C-terminalregion of the GPIIb heavy chain. Indigenous proteases, such as plasminor leukocyte enzymes, generated at the site of the thrombus in vivocleave the C-terminal portion of hGPIIb, releasing the small peptidefragments described above. Measurement of these fragments in vascularfluid or in urine provides a diagnostic correlate of the extent of thethrombotic process. These measurements are also valuable in monitoringthe course of thrombolysis induced by plasminogen activators.

[0034] PMI-1, a monoclonal antibody derived from immunization of micewith isolated human platelet plasma membranes, reacts with the carboxyterminal aspect of the heavy chain of the platelet membrane glycoproteinhGPIIb. Shadle et al., J. Cell Biol., 99:2056-2060 (1984); and Loftus etal., Proc. Natl. Acad. Sci. USA, 840:7114-7118 (1987). In the presentinvention, an enzyme-linked immunosorbent assay (ELISA) has beendeveloped to measure levels of PMI-1 antigenic material. The principalobservation, illustrated in FIG. 1, is that in vitro treatment ofplatelets with protease releases from the carboxy terminus of the GPIIbheavy chain a small (approximately 7 kDa, based on average MW of aminoacid residue =120) fragment that contains the PMI-1 epitope. Thefragment is referred to herein as the C-terminal hGPIIb fragment.

[0035] A potential site of cleavage of GPIIb-IIIa by indigeneousproteases is downstream from the cysteine residue at position 171 of thesequence shown in SEQ ID NO 1. The cysteine at position 171 participatesin disulfide bridging to the light chain of GPIIb. Calvette et al.,Biochem. J., 261:551-560 (1989). The preferred site for chymotrypsincleavage of GPIIb-IIIa when present on platelets is between residues 172and 173 of the sequence shown in SEQ ID NO 1.

[0036] The carboxy terminus of hGPIIb has been identified by Loftus etal., J. Biol. Chem. 263:11025 (1988). After initial processing to cleavethe GPIIb precursor protein into the heavy and light chain, alternateproteolytic processing results in partial trimming back of the carboxyterminus of hGPIIb. The partial proteolysis produces a polypeptidehaving either an lysine (K) or arginine (R) at the carboxy terminus,corresponding to residue 200 or 201, respectively, of the sequence shownin SEQ ID NO 1.

[0037] The capacity of the PMI-1 monoclonal antibody to inhibit plateletadhesion to collagen, Shadle et al., J. Cell Biol., 99:2056-2060 (1984),its upregulation by chelation of divalent cations, Ginsberg et al., J.Clin. Invest., 78:1103-1111 (1986), and by occupancy of GPIIb-IIIa withadhesive ligands, Frelinger et al., J. Biol. Chem., 263:12397-12402(1988), has been previously published by the applicants and is wellknown. There have, however, been no prior publications on the use of anELISA technique for detecting this novel hGPIIb fragment as a means ofidentifying and evaluating thrombosis.

[0038] As discussed before, and reviewed by Ginsberg et al., inImmunologic Analysis: Recent Progress in Diagnostic Labor. Immunol.,Nakamura et al., Eds. Masson Publishing, USA Inc., 199-209 (1982), therehave been numerous assays designed to quantitate released plateletgranule components in the circulation as a means for detecting plateletactivation. The present invention, however, does not involve release ofa granule component, but rather involves the detection of a proteolyticattack on a platelet surface component, and as such is less susceptibleto in vitro artifact, e.g., false positive results due to in vitroactivation of platelets.

[0039] III. hGPIIb Fragment and Analogs

[0040] A. Fragment

[0041] In one embodiment, the present invention contemplates asubstantially isolated C-terminal hGPIIb fragment having a molecularweight of about 3900 daltons, comprising an amino acid residue sequencethat corresponds to the carboxy-terminal portion of the amino acidresidue sequence of hGPIIb. The C-terminal hGPIIb fragment includes as aportion of its amino acid residue sequence the sequence represented bythe formula -IHPAHHK- shown is SEQ ID NO 1 from residue 194 to residue200, and has the capacity to immunoreact with the monoclonal antibody,PMI-1. Preferably, the C-terminal hGPIIb fragment has the amino acidresidue sequence shown in SEQ ID NO 1 from about residue 173 to aboutresidue 200.

[0042] A C-terminal hGPIIb fragment can be purified from human urine orplasma using well known immunoaffinity methods, such as described inExample 1c(2). Alternatively, C-terminal GPIIb fragment can be producedin vitro by proteolysis of platelets or by proteolysis of isolatedGPIIb-IIIa, and subsequently immunoaffinity purified as described inExample 1. Proteolysis of platelets or isolated GPIIb-IIIa using theprotease chymotrypsin is particularly preferred. The latter procedure ispreferred due to the availability of platelets and the amount ofGPIIb-IIIa contained therein.

[0043] C-terminal hGPIIb fragment is substantially isolated ifcompositions containing the fragment are comprised of greater than 5percent, by weight, of fragment per total protein in the composition.

[0044] C-terminal hGPIIb fragment, once isolated, is useful to produceimmunogen for raising antibody specific for the fragment, and for use inthe diagnostic methods and systems for detecting the presence and amountof C-terminal hGPIIb fragment in body fluid samples, as disclosedherein.

[0045] B. Polypeptides Analogs of the C-Terminal hGPIIb Fragment

[0046] One embodiment of the present invention contemplates apolypeptide that includes amino acid residue sequences capable ofmimicking antigenic determinants expressed by the C-terminal hGPIIbfragment. The hGPIIb fragment-mimicking polypeptide is also referred toherein as an analog, or hGPIIb analog. A hGPIIb polypeptide analog ofthe present invention comprises an amino acid residue sequence thatcorresponds, and preferably is identical, to a portion of the hGPIIbsequence shown in SEQ ID No 1 from residue 1 to residue 200. The hGPIIbanalog is from about 7 to about 200 amino acid residues in length,preferably less than about 50, and more preferably less than 20 residuesin length, and includes a minimum of about 7 contiguous residues of thehGPIIb amino acid residue sequence shown in SEQ ID NO 1 from residue 173to residue 200.

[0047] In one preferred embodiment, a hGPIIb analog includes at leastthe following amino acid residue sequence:

-IHPAHHK-,

[0048] representing the amino acid residues 194-200 shown in SEQ IDNO 1. In this embodiment, a hGPIIb analog is also capable ofimmunoreacting with the monoclonal antibody, PMI-1. The antibody PMI-1is known to be immunospecific for (i.e., immunoreacts with) the minimumepitope

-IHPAHHK-

[0049] on hGPIIb.

[0050] In another embodiment, a preferred hGPIIb analog is a polypeptidethat comprises an amino acid residue sequence corresponding to one ofthe following amino acid sequences: -PQPPVNPLK-,       -PLKVDWGLPIP-,              -PIPSPSPIHPAHHK-, or                      -IHPAHHK-,

[0051] having an amino acid residue sequence shown in SEQ ID NO 1 fromresidue 173 to residue 181, from residue 179 to residue 189, fromresidue 187 to residue 200, or from residue 194 to residue 200,respectively.

[0052] Particularly preferred hGPIIb analogs are contemplated having anamino acid residue sequence as shown in Table 1 below. TABLE 1Designation^(a) Amino Acid Residue Sequence p173-193PQPPVNPLKVDWGLPIPSPSP p173-181 PQPPVNPLK p179-189 PLKVDWGLPIP p187-200PIPSPSPIHPAHHK p194-200 IHPAHHK

[0053] One contemplated hGPIIb analog has an amino acid residue sequencethat corresponds to the sequence shown in SEQ ID NO 1 from residue 1 toresidue 200. More preferred is a hGPIIb analog that corresponds insequence to a C-terminal hGPIIb fragment and has a sequence shown in SEQID NO 1 from residue 173 to residue 200.

[0054] The polypeptides shown in Table 1 are further characterized bytheir ability to neutralize (competitively inhibit) the binding ofantibody molecules to their respective antigenic determinants on thenative hGPIIb molecule, described hereinbelow.

[0055] It should be understood that a hGPIIb analog of the presentinvention need not be identical to the amino acid residue sequence ofhGPIIb, so long as it has sufficient similarity with (i.e., correspondsto) portions of hGPIIb that the analog is able to competitively inhibitthe immunoreaction of specific antibody molecules, elicited as describedhereinbelow. Therefore, a hGPIIb analog can be subject to variouschanges, such as insertions, deletions and substitutions, eitherconservative or non-conservative, where such changes provide for certainadvantages in their use.

[0056] Conservative substitutions are those where one amino acid residueis replaced by another, biologically similar residue. Examples ofconservative substitutions include the substitution of one hydrophobicresidue, such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as betweenarginine and lysine, between glutamic and aspartic acids or betweenglutamine and asparagine and the like. The term “conservativesubstitution” also includes the use of a substituted amino acid in placeof an unsubstituted parent amino acid provided that such a polypeptidealso displays the requisite immunoreaction activity.

[0057] When a polypeptide of the present invention has a sequence thatis not identical to the corresponding sequence of hGPIIb because one ormore conservative or non-conservative substitutions have been made,usually no more than about 20% and preferably no more than 10% of theamino acid residues are substituted, except where additional residueshave been added at either terminus for the purpose of providing a“linker” by which the polypeptides of this invention can be convenientlyaffixed to a label or solid matrix, or antigenic carrier. Labels, solidmatrices and carriers that can be used with the polypeptides of thisinvention are described hereinafter.

[0058] Amino acid residue linkers are usually at least one residue andcan be 40 or more residues, more often 1 to 10 residues. Typical aminoacid residues used for linking are tyrosine, cysteine, lysine, glutamicand aspartic acid, or the like. In addition, a polypeptide sequence ofthis invention can differ from the natural sequence by the sequencebeing modified by terminal-NH₂ acylation, e.g., acetylation, orthioglycolic acid amidation, terminal-carboxlyamidation, e.g., ammonia,methylamine, etc.

[0059] When coupled to a carrier via a linker to form what is known inthe art as a carrier-hapten conjugate, a hGPIIb analog of the presentinvention is capable of inducing formation of antibodies thatimmunoreact with the C-terminal fragment of hGPIIb when present as afree-floating fragment in vascular fluid or urine.

[0060] In view of the well established principle of immunologiccross-reactivity, the present invention therefore contemplatesantigenically related variants of the polypeptides described before. An“antigenically related variant” is a polypeptide that is capable ofinducing formation of antibody molecules that immunoreact with a hGPIIbanalog and with the C-terminal hGPIIb fragment when present in bodyfluids.

[0061] Any hGPIIb analog of the present invention can be synthesized byone of many techniques known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, ease of production and the like. An excellent summary of themany techniques available can be found in J. M. Steward and J. D. Young,“Solid Phase Peptide Synthesis”, W. H. Freeman Co., San Francisco(1969); Bodansky et al., “Peptide Synthesis”, John Wiley & Sons, 2ndEd., (1976); and Meienhofer, “Hormonal Proteins and Peptides”, 2:46,Academic Press, NY (1983) for solid phase peptide synthesis; andSchroder et al., “The Peptides”, Vol. 1, Academic Press, NY (1965) forclassical solution synthesis, each of which is incorporated herein byreference. Appropriate protective groups usable in such synthesis aredescribed in the above texts and in McOmie, “Protective Groups inOrganic Chemistry”, Plenum Press, NY (1973), which is incorporatedherein by reference.

[0062] In general, the solid-phase synthesis methods contemplatedcomprise the sequential addition of one or more amino acid residues orsuitably protected amino acid residues to a growing peptide chain.Normally, either the amino or carboxyl group of the first amino acidresidue is protected by a suitable, selectively removable protectinggroup. A different, selectively removable protecting group is utilizedfor amino acids containing a reactive side group such as lysine.

[0063] Using a solid-phase synthesis as exemplary, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting groups (and solid support) are removedsequentially or concurrently, to afford the final polypeptide.

[0064] A hGPIIb analog can be used, inter alia, in the diagnosticmethods and systems of the present invention to detect C-terminal hGPIIbfragment present in a body sample, or can be used to prepare an inoculumas described herein for the preparation of antibodies that immunoreactwith the fragment.

[0065] IV. Antibodies and Antibody Compositions

[0066] The term “antibody” in its various grammatical forms is usedherein to refer to immunoglobulin molecules and fragments thereof,namely immunologically active portions of immunoglobulin molecules,i.e., molecules that contain an antibody combining site or paratope.Illustrative antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v).

[0067] The term “antibody combining site” refers to that structuralportion of an antibody molecule, comprised of a heavy and light chainvariable and hypervariable regions, that specifically binds(immunoreacts with) an antigen. The term “immunoreact” in its variousforms is used herein to refer to binding between an antigenicdeterminant-containing molecule and a molecule containing an antibodycombining site such as a whole antibody molecule or a portion thereof.

[0068] The term “antigenic determinants” refers to the actual structuralportion of the antigen that is immunologically bound by an antibodycombining site. The term is also used interchangeably with “epitope”.

[0069] As used herein, the term “specifically bound” refers to anon-random binding reaction between an antibody and a antigen.

[0070] A. Antibody Compositions

[0071] An antibody composition of the present invention is characterizedas containing antibody molecules that immunoreact with a C-terminalhGPIIb fragment or hGPIIb analog as described herein.

[0072] An antibody of this invention, i.e., an anti-C-terminal hGPIIbfragment antibody, does not immunoreact with other portions of thehGPIIb molecule, such as antigenic determinants defined by polypeptidescorresponding in sequence to regions of hGPIIb that do not includeresidues 173 through 200 of the sequence shown in SEQ ID NO 1. Thus, forexample, a contemplated antibody does not immunoreact with a polypeptidehaving a sequence represented by the formula REQNSLDSWGPK, shown in SEQID NO 1 from residue 113 to residue 124. That is, the antibody isspecific for the C-terminal hGPIIb fragment identified and definedherein.

[0073] Thus an antibody, in its various embodiments, immunoreacts withan epitope present on a C-terminal hGPIIb fragment. Exemplary epitopesare defined by the C-terminal hGPIIb analogs designated p173-181,p179-189, p187-200 or p194-200. Additional epitopes can reside withinthe hGPIIb C-terminal fragment that are useful for defining theimmunoreactivity of an antibody of this invention. Thus otherembodiments contemplate an antibody that immunoreacts with a polypeptidedefining regions other than the p194-200 portion of hGPIIb C-terminalfragment. Exemplary antibodies immunoreact with the polypeptidep173-193, shown in SEQ ID NO 1 from residues 173 to 193.

[0074] Such a contemplated antibody composition is typically produced byimmunizing a mammal with an inoculum containing a C-terminal hGPIIbfragment of this invention, or a polypeptide analog, thereby inducingproduction in the mammal of antibody molecules having immunospecificityfor the C-terminal hGPIIb fragment. The antibody molecules are thencollected from the mammal and isolated to the extent desired by wellknown techniques such as, for example, by using DEAE Sephadex to obtainthe IgG fraction. To enhance the specificity of the antibody, theantibodies may be purified by immunoaffinity chromatography using solidphase-affixed immunizing polypeptide or C-terminal hGPIIb fragment. Theantibody is contacted with the solid phase-affixed immunizingpolypeptide or fragment (immunogen) for a period of time sufficient forthe immunogen to immunoreact with the antibody molecules to form a solidphase-affixed immunocomplex. The bound antibodies are separated from thecomplex by standard techniques.

[0075] Preferred and exemplary are the methods to produce and isolateantibodies described in Example 3.

[0076] The antibody composition so produced can be used inter alia, inthe diagnostic methods and systems of the present invention to detectthe presence and/or amount of C-terminal hGPIIb fragment in a bodysample.

[0077] A preferred antibody in an antibody composition of this inventionis a monoclonal antibody.

[0078] B. Inocula

[0079] The word “inoculum” in its various grammatical forms is usedherein to describe a composition containing a polypeptide or theC-terminal hGPIIb fragment of this invention as an active ingredientused for the preparation of antibodies that immunoreact with theC-terminal hGPIIb fragment. The inoculum also contains apharmaceutically acceptable aqueous diluent such that, when administeredby immunization, is capable of eliciting antibodies that immunoreactwith the hGPIIb C-terminal fragment.

[0080] When a polypeptide is used to induce antibodies it is to beunderstood that the polypeptide can be used alone, or linked to acarrier as a conjugate, or as a polypeptide polymer, but for ease ofexpression, the various embodiments of the polypeptides of thisinvention are collectively referred to herein by the term “polypeptide”,and its various grammatical forms.

[0081] For a polypeptide that contains fewer than about 35 amino acidresidues, it is preferable to use the peptide bound to a carrier for thepurpose of inducing the production of antibodies as already noted.

[0082] As discussed before, one or more additional amino acid residuescan be added to the amino- or carboxy-termini of the polypeptide toassist in binding the polypeptide to a carrier. Cysteine residues addedto the amino- or carboxy-termini of the polypeptide have been found tobe particularly useful for forming conjugates via disulfide bonds.However, other methods well known in the art for preparing conjugatescan also be used. Exemplary additional linking procedures include theuse of Michael addition reaction products, di-aldehydes such asglutaraldehyde, Klipstein et al., J. Infect. Dis., 147, 318-326 (1983)and the like, or the use of carbodiimide technology, as in the use of awater-soluble carbodiimide to form amide links to the carrier. For areview of protein conjugation or coupling through activated functionalgroups, see Avrameas, et al., Scand. J. Immunol., 1:7-23 (1978).

[0083] Useful carriers are well known in the art, and are usuallyproteins themselves. Examples of such carriers are keyhole limpethemocyanin (KLH), edestin, thyroglobulin, albumins, such as bovine serumalbumin (BSA) or human serum albumin (HSA), red blood cells, such assheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well aspolyamino acids, such as poly (D-lysine: D-glutamic acid), and the like.

[0084] The choice of carrier is dependent upon the ultimate use of theinoculum and is based upon criteria not particularly involved in thepresent invention. For example, a carrier that does not generate anundesired reaction in the particular animal to be inoculated should beselected.

[0085] The inoculum contemplated herein contains an effectiveimmunogenic amount of an immunogen of this invention, e.g., an hGPIIbanalog or a C-terminal hGPIIb fragment, typically as a conjugate linkedto a carrier. The effective amount of polypeptide or protein per unitdose sufficient to induce an immune response to the immunogen depends,among other things, on the species of animal inoculated, the body weightof the animal and the chosen inoculation regimen as is well known in theart. Inocula typically contain polypeptide or protein concentrations ofabout 10 micrograms to about 500 milligrams per inoculation (dose),preferably about 50 micrograms to about 50 milligrams per dose.

[0086] The term “unit dose” as it pertains to the inocula of the presentinvention refers to physically discrete units suitable as unitarydosages for animals, each unit containing a predetermined quantity ofactive material calculated to produce the desired immunogenic effect inassociation with the required diluent; i.e., carrier, or vehicle. Thespecifications for the novel unit dose of an inoculum of this inventionare dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular immunologiceffect to be achieved, and (b) the limitations inherent in the art ofcompounding such active material for immunologic use in animals, asdisclosed in detail herein, these being features of the presentinvention.

[0087] Inocula are typically prepared from the dried solidpolypeptide-conjugate by dispersing the polypeptide-conjugate in aphysiologically tolerable (acceptable) diluent or vehicle such as water,saline or phosphate-buffered saline to form an aqueous composition. Suchdiluents are well known in the art and are discussed, for example, inRemington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company,Easton, Pa. (1980) at pages 1465-1467.

[0088] Inocula can also include an adjuvant as part of the diluent.Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund'sadjuvant (IFA) and alum are materials well known in the art, and areavailable commercially from several sources.

[0089] C. Monoclonal Antibody Compositions

[0090] The phrase “monoclonal antibody composition” in its variousgrammatical forms refers to a population of antibody molecules thatcontain only one species of antibody combining site capable ofimmunoreacting with a particular antigen. A monoclonal antibodycomposition thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody composition istypically composed of antibodies produced by clones of a single cellcalled a hybridoma that secretes (produces) but one kind of antibodymolecule. The hybridoma cell is formed by fusing an antibody-producingcell and a myeloma or other self-perpetuating cell line. Such antibodieswere first described by Kohler and Milstein, Nature 256:495-497 (1975),which description is incorporated by reference. Detailed preparation ofa monoclonal antibody composition is described further herein.

[0091] Thus, a monoclonal antibody of this invention immunoreacts with aC-terminal hGPIIb fragment or hGPIIb analog of this invention, but doesnot substantially immunoreact with a polypeptide having a sequencerepresented by the formula REQNSLDSWGPK shown in SEQ ID NO 1 fromresidue 113 to residue 124. That is, the antibody is specific for theC-terminal hGPIIb fragment.

[0092] Particularly preferred is a monoclonal antibody that immunoreactswith a hGPIIb analog of this invention, particularly the polypeptideIHPAHHK or polypeptides that contain the sequence

-IHPAHHK-

[0093] shown in SEQ ID NO 1 from residues 194 to 200. Exemplary of thispreferred embodiment is the monoclonal antibody PMI-1, available fromthe American Type Culture Collection (ATCC, Rockville, Md.) and havingATCC accession number HB 9476, that has been shown to be immunospecificfor an epitope on hGPIIb defined by the polypeptide IHPAHHK and presenton a C-terminal hGPIIb fragment.

[0094] In a related embodiment a monoclonal antibody is contemplatedthat immunoreacts with a hGPIIb analog defined by a polypeptidecomprising a sequence of amino acid residues shown in SEQ ID NO 1 fromresidues 173 to 193, particularly a polypeptide having the sequenceshown in SEQ ID NO 1 from residues 173 to 181, from residues 179 to 189,or from residues 179 to 193.

[0095] V. Methods for Producing Monoclonal Antibody Compositions

[0096] The present invention contemplates a method of forming amonoclonal antibody molecule that (a) immunoreacts with an antigenicdeterminant (epitope) on the C-terminal hGPIIb fragment, said fragmentincluding an amino acid residue sequence as shown in SEQ ID NO 1 fromresidues 173 to 200, from residues 173 to 181, from residues 173 to 193,from residues 179 to 189, from residues 179 to 193, or from residues 194to 200, but (b) does not substantially immunoreact with a polypeptiderepresented by the formula REQNSLDSWGPK shown in SEQ ID NO 1 fromresidues 113 to 124.

[0097] A preferred method comprises the steps of:

[0098] (a) Immunizing an animal with an inoculum containing a C-terminalhGPIIb fragment or a polypeptide hGPIIb analog as described herein asthe immunogen. Preferably, the immunogen is complexed with a carrier orimmunopotentiator such as an ISCOM particle, liposome or the like. Theinoculum is administered to an immunologically competent mammal in anamount sufficient to produce an immune response. Preferably, the mammalis a rodent such as a rabbit, rat or mouse. The mammal is thenmaintained for a time period sufficient for it to produce cellssecreting antibody molecules that immunoreact with the C-terminal hGPIIbfragment.

[0099] (b) Preparing a suspension of immune cells. This is typicallyaccomplished by removing the spleen of the mammal and mechanicallyseparating the individual spleen cells in a physiologically tolerablemedium using methods well known in the art.

[0100] (c) Treating the suspended antibody-producing cells with atransforming agent capable of producing a transformed (“immortalized”)cell line. Transforming agents and their use to produce immortalizedcell lines are well known in the art and include DNA viruses such asEpstein Bar Virus (EBV), Simian Virus 40 (SV40), Polyoma Virus and thelike, RNA viruses such as Moloney Murine Leukemia Virus (Mo-MuLV), RousSarcoma Virus and the like, myeloma cells such as P3X63-Ag8.653,Sp2/O-Ag14, that are available from the ATCC under the designations CRL1580 and CRL 1581, respectively, and the like.

[0101] In preferred embodiments, treatment with the transforming agentresults in the production of a hybridoma by means of fusing thesuspended spleen cells with mouse myeloma cells from a suitable cellline by the use of a suitable fusion promoter. The preferred ratio isabout 5 spleen cells per myeloma cell in a suspension containing about10⁸ splenocytes. A preferred fusion promoter is polyethylene glycolhaving an average molecule weight from about 1000 to about 4000(commercially available as PEG 1000, etc.); however, other fusionpromoters known in the art can be employed.

[0102] The cell line used should preferably be of the so-called “drugresistant” type, so that unused myeloma cells will die in a selectivemedium, whereas hybrids will survive. The most cell lines used for thispurpose are 8-azaguanine resistant. These lines lack the enzymehypoxanthine-guanine phosphoribosyl transferase and hence will not besupported by HAT (hypoxanthine, aminopterin, and thymidine) medium. Itis also generally preferred that the myeloma cell line used be of theso-called “non-secreting” type, i.e., does not itself produce antibodymolecules. In certain cases, however, secreting myeloma lines may bepreferred.

[0103] (d) Cloning the transformed cells, preferably to monoclonality.The cloning is preferably performed in a tissue culture medium that willnot sustain (support) non-transformed cells. When the transformed cellsare hybridomas, this is typically performed by diluting and culturing inseparate containers the mixture of unused spleen cells, unused myelomacells, and fused cells (hybridomas) in a selective medium which will notsustain the unused myeloma cells. The cells are cultured in this mediumfor a time sufficient to cause death of the unused cells (about oneweek). The dilution can be a limiting dilution, in which the volume ofdiluent is statistically calculated to isolate a certain number of cells(e.g., 1-4) in each separate container (e.g., each well of a microtiterplate). The medium is one (e.g., HAT medium) that will not sustain thedrug-resistant (e.g., 8-azaguanine resistant) unused myeloma cell line.

[0104] (e) Screening the tissue culture medium of the clonedtransformants to detect the presence of secreted antibody molecules asdiscussed in Example 4a. Preferably, the medium is monitored using wellknown immunological techniques for the appearance of antibody moleculesthat immunoreact with C-terminal hGPIIb fragment.

[0105] (f) Selecting a desired transformant and growing it in anappropriate tissue culture medium for a suitable length of time, andharvesting the antibody from the culture supernatant by well knowntechniques. The suitable medium and suitable length of culturing timeare also well known or are readily determined.

[0106] A monoclonal antibody of the present invention can also beproduced by preparing a culture containing a monoclonal hybridoma thatsecretes antibody molecules of the appropriate polypeptide specificity.The culture is maintained under conditions and for a time periodsufficient for the hybridoma to secrete the antibody molecules into themedium. The antibody-containing medium is then collected. The antibodymolecules can then be further isolated by well known techniques.

[0107] Representative and preferred methods for producing monoclonalantibody compositions of this invention are described in Example 3c.

[0108] To produce a much greater concentration of slightly less puremonoclonal antibody, the selected hybridoma can be transferred byinjection into mice, preferably syngeneic or semisyngeneic mice. Thehybridoma will cause formation of antibody-producing ascites tumorsafter a suitable incubation time, which will result in a highconcentration of the desired antibody (about 5-20 mg/ml) in thebloodstream and peritoneal exudate of the host mouse.

[0109] Media and animals useful for the preparation of thesecompositions are both well known in the art and commercially availableand include synthetic culture media, inbred mice and the like. Onecommonly used synthetic medium is Dulbecco's minimal essential medium[DMEM; Dulbecco et al., Virol. 8:396 (1959)] supplemented with 4.5 gm/lglucose, 20 mM glutamine, and 20% fetal calf serum. A typical inbredmouse strain is the Balb/c.

[0110] The monoclonal antibody compositions produced by the above methodcan be used in the same manner as disclosed herein for antibodies ofthis invention, and are preferred.

[0111] For example, the monoclonal antibody can be used in thediagnostic methods and systems disclosed herein where formation of aC-terminal hGPIIb fragment-containing immunoreaction product is desired.

[0112] VI. Hybridomas

[0113] Hybridomas of the present invention are those characterized ashaving the capacity to produce an anti-GPIIb C-terminal fragmentmonoclonal antibody as described before.

[0114] Representative preferred hybridomas are prepared as described inExample 3. Particularly preferred is the hybridoma culture designatedPMI-1.

[0115] Hybridoma culture PMI-1 has been deposited pursuant to BudapestTreaty requirements with the American Type Culture Collection (ATCC),Rockville, Md., on May 17, 1989, and was assigned accession number HB9476.

[0116] Hybridoma HB 9476 was deposited in a depository affordingpermanence of the deposit and ready accessibility thereto by the publicupon the issuance of a patent, under conditions which assure that accessto the hybridoma will be available during the pending of the patentapplication to those deemed by the Commissioner to be entitled to suchaccess, and that all restrictions on the availability to the public ofthe hybridoma as deposited will be irrevocably removed upon the grantingof the patent. The deposited hybridoma will be maintained by the ATCCfor the term of the patent or 30 years from the date of deposit,whichever is longer, and in all events for at least five years after thedate of the last request for access. All maintenance fees have beenpaid.

[0117] Methods for producing hybridomas secreting antibody moleculeshaving a desired immunospecificity, i.e., having the ability toimmunoreact with a particular protein, an identifiable epitope on aparticular protein and/or a polypeptide, are well known in the art andare described further herein. Particularly applicable is the hybridomatechnology described by Niman et al., Proc. Natl. Acad. Sci. USA,80:4949-4953 (1983), and by Galfre et al., Meth. ; Enzymol., 73:3-46(1981), which descriptions are incorporated herein by reference.

[0118] Other methods of producing a monoclonal antibody, a hybridomacell, or a hybridoma cell culture are also well known. See, for example,the method of isolating monoclonal antibodies from an immunologicalrepertoire as described by Sastry, et al., Proc. Natl. Acad. Sci.,86:5728-5732 (1989); and Huse et al., Science, 246:1275-1281 (1989).

[0119] Also contemplated by this invention is the hybridoma cell, andcultures containing a hybridoma cell that produce a monoclonal antibodyof this invention.

[0120] VII. Diagnostic Systems

[0121] A diagnostic system of the present invention in kit formincludes, in an amount sufficient to perform at least one assay, acomposition containing polyclonal or monoclonal antibody of thisinvention or fragments thereof, as a separately packaged reagent.Instructions for use of the packaged reagent are also typicallyincluded.

[0122] “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like. Also included, in one formor another, may be charts, graphs and the like that demonstratepredetermined concentration levels correlating specific physiologicalconditions to thrombotic events.

[0123] A diagnostic system is contemplated for assaying for thepresence, and preferably amount, of C-terminal hGPIIb fragment in a bodyfluid sample, such as blood, plasma or urine according to the diagnosticmethods described herein.

[0124] Preferably, the antibody is present as a monoclonal antibodycomposition, comprising a monoclonal antibody as described herein.

[0125] A diagnostic system of the present invention typically alsoincludes a label or indicating means capable of signaling the formationof a specifically bound complex containing an antibody molecule of thepresent invention.

[0126] As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an antibody molecule that ispart of an antibody or monoclonal antibody composition of the presentinvention, or used separately, and those atoms or molecules can be usedalone or in conjunction with additional reagents. Such labels arethemselves well-known in clinical diagnostic chemistry and constitute apart of this invention only insofar as they are utilized with otherwisenovel methods and/or systems.

[0127] The labeling means can be a fluorescent labeling agent thatchemically binds to antibodies or antigens without denaturing them toform a fluorochrome (dye) that is a useful immunofluorescent tracer.Suitable fluorescent labeling agents are fluorochromes such asfluorescein isocyanate (FIC), fluorescein iosthiocyante (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca“Immunofluorescence Analysis”, in Antibody As A Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982) which isincorporated herein by reference.

[0128] In preferred embodiments, the indicating group is an enzyme, suchas horseradish peroxidase (HRO), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

[0129] Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabelling agent is aradioactive element that produces gamma ray emissions. Elements whichthemselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Crrepresent one class of gamma ray emission-producing radioactive elementindicating groups. Particularly preferred is ¹²⁵I. Another group ofuseful labelling means are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³Nwhich themselves emit positrons. The positrons so emitted produce gammarays upon encounters with electrons present in the animal's body. Alsouseful is a beta emitter, such as ¹¹¹indium of ³H.

[0130] The linking of labels, i.e., labeling of, polypeptides andproteins is well known in the art. For instance, antibody moleculesproduced by a hybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Avrameas, et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

[0131] The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention but is not itself an antibody molecule of the presentinvention. Exemplary specific binding agents are antibody molecules,complement proteins or fragments thereof, protein A and the like.Preferably, the specific binding agent can bind the antibody molecule ofthis invention when it is present as part of a complex.

[0132] In preferred embodiments the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

[0133] The diagnostic kits of the present invention can be used in an“ELISA” format to detect, for example, the presence or quantity ofC-terminal hGPIIb fragment in a body fluid sample such as serum, plasmaor urine. “ELISA” refers to an enzyme-linked immunosorbent assay thatemploys an antibody or antigen bound to a solid phase and anenzyme-antigen or enzyme-antibody conjugate to detect and quantify theamount of an antigen present in a sample. A description of the ELISAtechnique is found in Chapter 22 of the 4th Edition of Basic andClinical Immunology by D. P. Sites et al., published by Lange MedicalPublications of Los Altos, Calif. in 1982 and in U.S. Pat. No.3,654,090; 3,850,752; and . 4,016,043, which are all incorporated hereinby reference. An exemplary ELISA protocol is described in Example 4.

[0134] Thus, in preferred embodiments, the antibody or antigen reagentcomponent can be affixed (operatively linked) to a solid matrix to forma solid support that is separately packaged in the subject diagnosticsystems. The reagent is typically affixed to the solid matrix byadsorption from an aqueous medium, although other modes of affixation,well known to those skilled in the art, can be used. Exemplaryaffixation methods are described in Example 4.

[0135] Particularly preferred are embodiments suitable for competitionELISA assays wherein the antibody is in the liquid phase together with asample containing an unknown amount of C-terminal hGPIIb fragment andthe antigen is in the solid phase in an amount sufficient to competewith liquid phase antigen for immunoreaction with the liquid phaseantibody. In this embodiment, which is described in Example 4, theantigen can be hGPIIb analog, C-terminal hGPIIb fragment, or isolatedGPIIb-IIIa.

[0136] Useful solid matrices are well known in the art. Such materialsinclude the cross-linked dextran available under the trademark SEPHADEXfrom Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; polystyrenebeads about 1 micron to about 5 millimeters in diameter available fromAbbott Laboratories of North Chicago, Ill.; polyvinyl chloride,polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-basedwebs such as sheets, strips or paddles; or tubes, plates or the wells ofa microtiter plate such as those made from polystyrene orpolyvinylchloride.

[0137] Thus, in particularly preferred embodiments, a diagnostic kitfurther contains, in a separate package, an antigen as described abovefor use in a the competitive ELISA assay in the form of a solid phaseantigen as defined above.

[0138] The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

[0139] The packages discussed herein in relation to diagnostic systemsare those customarily utilized in diagnostic systems. Such packagesinclude glass and plastic (e.g., polyethylene, polypropylene andpolycarbonate) bottles, vials, plastic and plastic-foil laminatedenvelopes and the like.

[0140] VIII. Assay Methods

[0141] The present invention contemplates any diagnostic method thatresults in detecting C-terminal hGPIIb fragment in a body fluid sampleusing an hGPIIb analog, a substantially isolated C-terminal hGPIIbfragment or an antibody of this invention as a reagent to form animmunoreaction product whose amount relates, either directly orindirectly, to the presence, and preferably amount, of C-terminal hGPIIbfragment in the sample.

[0142] Those skilled in the art will understand that there are numerouswell known clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product whose amount relates to the amount of C-terminalhGPIIb fragment present in a body sample. Thus, while exemplary assaymethods are described herein, the invention is not so limited.

[0143] Various heterogeneous and homogeneous protocols, eithercompetitive or noncompetitive, can be employed in performing an assaymethod of this invention.

[0144] Generally, to detect the presence of a C-terminal hGPIIb fragmentin a patient, an aliquot (i.e., a predetermined amount) of a body fluidsample, such as urine or a vascular fluid, namely blood, plasma or serumfrom the patient is contacted by admixture (admixed), with an antibodycomposition of the present invention to form an immunoreactionadmixture. The admixture is then maintained under biological assayconditions for a period of time sufficient for the C-terminal hGPIIbfragment present in the sample to immunoreact with (immunologicallybind) a portion of the antibody combining sites present in the antibodycomposition to form a C-terminal hGPIIb fragment-antibody moleculeimmunoreaction product (immunocomplex). The complex can then be detectedas described herein. The presence of the complex is indicative ofC-terminal hGPIIb fragment in the sample.

[0145] Maintenance time periods sufficient for immunoreaction are wellknown and are typically from about 10 minutes to about 16-20 hours at atemperature of about 4C. to about 45C., with the time and temperaturetypically being inversely related. For example, longer maintenance timesare utilized at lower temperatures, such as 16 hours at 4C., and shortertimes for higher temperatures, such as 1 hour at room temperature.

[0146] Biological assay conditions are those that maintain thebiological activity of the immunochemical reagents of this invention andthe C-terminal hGPIIb fragment sought to be assayed such that thereagents retain their ability to form an immunoreaction product. Thoseconditions include a temperature range of about 4C. to about 45C., a pHvalue of about 5 to about 9 and an ionic strength varying from that ofdistilled water to that of about one molar sodium chloride. Methods foroptimizing such maintenance time periods and biological assay conditionsare well known in the art.

[0147] Preferred maintenance times and assay conditions sufficient forimmunoreaction to occur are described in Example 4.

[0148] According to the discoveries described herein, thrombosis isaccompanied by the cleavage of the carboxy terminus of hGPIIb byindigenous proteases, to form soluble C-terminal hGPIIb fragment in bodyfluid samples. Thus, in one embodiment, the detection of C-terminalhGPIIb fragment in a body sample is utilized as a means to monitor thepresence of an in vivo thrombic event in a patient (thrombosis) using anantibody molecule specific for the C-terminal hGPIIb fragment asdisclosed herein.

[0149] In preferred embodiments, immunoassay of C-terminal hGPIIbfragment is conducted using a cell free body fluid sample. By cell freeis meant that the sample does not contain detectable amounts of cells,tissue or other macroscopic biological materials normally present in abody fluid such as blood. A body fluid sample typically contains cellsas a normal component, or as a contaminant, and can be renderedcell-free by a variety of biochemical procedures includingcentrifugation, filtration and chromatography, so long as the retainedfluid sample contains substantially all of the soluble protein initiallypresent in the sample prior to removal of the cells. Particularlypreferred are centrifugation steps such as described in Example 4.

[0150] Thus a body fluid sample, such as blood, known to contain bothplatelets having GPIIb-IIIa thereon and soluble C-terminal hGPIIbfragment, is preferably treated, prior to admixing, to a manipulationthat results in forming a cell-free body fluid sample, ie, separatingthe cells present in the sample away from the body fluid sample to forma cell-free body fluid sample. An aliquot of the cell-free body fluidsample is then admixed with an antibody of the invention to form animmunoreaction admixture.

[0151] Alternatively, the separation of C-terminal hGPIIb fragment fromcells having GPIIb, such as platelets, may be accomplished at otherstages of the assay methods disclosed herein. For example, theimmunoreaction product containing C-terminal hGPIIb fragment can beseparated from platelets present in the body fluid sample bycentrifugation after the maintenance step under conditions that separatecells, i.e., platelets, from protein complexes such as an immunocomplexbetween C-terminal hGPIIb fragment and an antibody of this invention.

[0152] Determining the presence or amount of C-terminal hGPIIbfragment-containing immunoreaction product formed by the abovemaintenance step, either directly or indirectly, can be accomplished byassay techniques well known in the art, and typically depend on the typeof indicating means used.

[0153] In a preferred competition assay method, the immunoreactionadmixture described above further contains a solid phase having affixedthereto a solid phase antigen comprising a hGPIIb analog or apolypeptide having an amino acid residue sequence that includes thesequence of a C-terminal hGPIIb fragment of this invention. Thus, inthis embodiment, the assay comprises the steps of:

[0154] (a) admixing a body fluid sample with 1) an antibody compositionof this invention and 2) a solid support having affixed thereto(operatively linked) a hGPIIb analog or a polypeptide having an aminoacid residue sequence that includes the sequence of a C-terminal hGPIIbfragment of this invention, or both, to form an immunoreaction admixturehaving both a liquid phase and a solid phase;

[0155] (b) maintaining said immunoreaction admixture under biologicalassay conditions for a time period sufficient to form an immunoreactionproduct in the solid phase; and

[0156] (c) detecting the presence, and preferably amount, of theimmunoreaction product formed in the solid phase in step (b), andthereby the amount of presence/amount of C-terminal hGPIIb fragment inthe body fluid sample.

[0157] Where an hGPIIb analog is used in the solid phase, the antibodycomposition contains antibody molecules that immunoreact with the hGPIIbanalog. Preferably, the solid phase antigen is isolated GPIIb-IIIa,prepared as described in Example 1b, or substantially isolatedC-terminal hGPIIb fragment, prepared as described in Example 1c.

[0158] Preferably, the body fluid sample is a cell free body fluidsample such as urine or platelet poor plasma.

[0159] More preferably, detecting in step (c) is performed by the stepsof:

[0160] (i) admixing the immunoreaction product formed in step (b) withan indicating means to form a second reaction admixture;

[0161] (ii) maintaining the second reaction admixture for a time periodsufficient for said indication means to bind the immunoreaction productformed in step (b) and form a second reaction product; and,

[0162] (iii) determining the presence and/or amount of indicating meansin the second reaction product, and thereby the presence of theimmunoreaction product formed in step (b). Particularly preferred is theuse of a labeled second antibody, immunospecific for the first antibody,as the indicating means, and preferably the label is horseradishperoxidase. In one embodiment, it is particularly preferred to use (1)isolated GPIIb-IIIa as the solid phase antigen, (2) PMI-1 monoclonalantibody in the antibody composition, and (3) goat anti-mouse IgGantibodies labeled with horseradish peroxidase as the indicating means.Exemplary is the competition ELISA format described in Example 4.

[0163] In another competition assay format the immunoreaction admixturecontains (1) a body fluid sample, preferably cell free, (2) an antibodyof this invention and (3) a labeled hGPIIb analog or labeled C-terminalhGPIIb fragment, wherein the antibody is present in the solid phase,being affixed to a solid support, to form a liquid and a solid phase. Inthis embodiment, the admixed body fluid sample competes with the labeledreagent for immunoreaction with the solid phase antibody to form a solidphase immunoreaction product. Thereafter, the detection of label in thesolid phase correlates with the amount of C-terminal hGPIIb fragment inthe admixed fluid sample.

[0164] The following examples are intended to illustrate, but not limit,the present invention.

EXAMPLE

[0165] 1. Preparation of Substantially Isolated C-Terminal hGPIIbFragment

[0166] a. Platelet Isolation

[0167] One unit of whole human blood was collected in ACD (0.065 Mcitric acid, 0.085 M sodium citrate, 2% dextrose) containing hirudin(Sigma Chemical Co., St. Louis, Mo.) at a final concentration of 0.06units per milliliter (U/ml) and centrifuged for 15 minutes (min) at120×g. The resulting supernatant, designated platelet-rich plasma (PRP),was recovered, isolated and further centrifuged for 15 min at 1200×g toform a pellet of isolated platelets. The supernatant formed wascollected and designated platelet-poor plasma (PPP).

[0168] b. GPIIb-IIIa Isolation from Platelets

[0169] A platelet pellet prepared as in Example 1a, was resuspended in 5ml TBS (0.15 M NaCl, 0.2 M Tris, pH 7.4, 5×10⁻⁴ M CaCl₂, 10⁻⁵ Mleupeptin) and sonicated on ice for 10 min at a maximum setting using aModel W-375 sonicator (Heat Systems Ultrasonics, Plainview, N.Y.). Thesonicated suspension was twice frozen and thawed using a dryice-methanol ice bath and stored at minus 20 degrees C (20 C.). Thefrozen-thawed platelet sonicate was layered on top of 5 ml of a sucrosesolution (40% v/v in TBS), and centrifuged at 4 degrees C. for one hourat 38,000 rotations per minute (RPM) in a SW41 centrifuge rotor (BeckmanInstruments, Fullerton, Calif.) to form a milky colored infranatant. Themilky-infranatant was then recovered and centrifuged at 43,000 RPM in aSW50.1 centrifuge rotor (Beckman) at 4 C. for one hour. The resultingpellet was resuspended in typically 1-2 ml TBS to form a plateletmembrane solution, the protein concentration of which was determined tobe in the range of 10-25 mg/ml, typically using the Bio-Rad ProteinAssay Kit (Bio-Rad, Richmond, Calif.) according to the manufacturer'sinstructions.

[0170] The platelet membrane solution was again centrifuged in a SW50.1centrifuge rotor as above and the resulting pellet was resuspended in 2ml of extraction buffer (0.03 M Tris, pH 7.4, 1×10⁻⁵ M leupeptin, 200 mMn-octyl-beta-D-glucopyranoside; Calbiochem-Behring, La Jolla, Calif.).The platelet membrane extract thus formed was admixed thoroughly byvortexing and then maintained at room temperature for 30 min. Theextract was thereafter centrifuged at 45,000 rpm in a SW50.1 centrifugerotor for 1 hour at 4 C. and the platelet membrane extract supernatantthus formed was recovered.

[0171] The recovered supernatant was applied to a LKB Ultrogel Aca 34gel filtration column (3×97 cm, LKB Instruments, Gaithersburg, Md.) thathad been equilibrated with 1 liter of column buffer (0.03 M Tris, pH7.4, 0.1 mM CaCl₂, 0.1% n-octyl-beta-D-glucopyranoside) and 5 mlfractions were collected from the resulting column effluent. The opticaldensity at 280 nanometers of each fraction was determined and fractionsaround the several peaks were combined to form a pool for each peak.Samples from each pool were analyzed by electrophoresis in 6%polyacrylamide slab gels using the reducing buffers and proceduresdescribed by Laemmli, Nature (London), 227:680-685 (1970), and lowmolecular weight protein standards ranging in size from 14.4 kilodaltons(kDa) to 92.5 kDa (Bio-Rad, Richmond, Calif.). The pool containingpredominantly two protein species having molecular weights correspondingto GPIIb and GPIIIa, i.e., 120 kDa and 100 kDa, respectively wasrecovered and is designated as a composition containing isolatedGPIIb-IIIa. The protein concentration of the isolated GPIIb-IIIacontaining-composition thus prepared was typically determined using theBio-Rad Protein Assay Kits to be in the range of 0.3 to 0.8 mg/ml.

[0172] c. Isolation of Carboxy-Terminal Fragment of hGPIIb fromPlatelets

[0173] (1) Digestion with Proteases

[0174] A platelet pellet prepared as in Example 1a was resuspended inTyrode's solution (Sigma; 137 mM NaCl, 2 mM KCl, 12 mM NaHCO₃, 0.3 mMNaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 5.5 mM glucose, 5 mM HEPES, pH 7.4)supplemented with 100 nM PGE1 (Sigma; prostaglandin inhibitor ofactivation), apyrase (Sigma)(primary suspension contained 7.5 ug/ml ofPRP; washes contained 25 ug/ml), and BSA, 3.5 mg/ml. After twocentrifugations in supplemented Tyrode's, the platelet concentration wasadjusted to 1×10⁹ per ml in supplemented Tyrode's. Alpha-chymotrypsin(Sigma) was added to produce a final concentration of 0.75 mg/ml and thesolution was incubated for 60 min at 37 degrees C. The reaction was thenstopped by adding a 200 mM stock solution of phenylmethylsulfonylfluoride (PMSF) to form a final concentration of 1.0 mM PMSF in theplatelet suspension.

[0175] During the time course of the 1 hour chymotrypsin incubation ofthe platelet suspension, aliquots of 0.2 ml containing 2×10⁸ plateletswere taken and processed further. A first aliquot (0.2 ml) of plateletsuspension was centrifuged at 15,000×g for 5 min at room temperature toform a supernatant and a pellet. The supernatant was collected to form asupernatant sample. The pellet was resuspended in 0.2 ml of supplementedTyrode's containing 1 mM PMSF to form a resuspended pellet sample foruse in the ELISA assay described in Example 4b. A second aliquot (0.2ml) of platelet suspension was used directly as a total sample in theELISA assay of Example 4b. The presence, if any, of C-terminal hGPIIbfragment was then measured in the three samples (supernatant sample,resuspended pellet sample, and total sample extract) using thecompetition ELISA assay described in Example 4b.

[0176] The results are shown in FIG. 1, and indicate the levels of thePMI-1 epitope (FIG. 1A) and the levels of the TAB epitope (FIG. 1B) inthe three fractions. Monoclonal antibody PMI-1 immunoreacts with thePMI-1 epitope on hGPIIb defined by the polypeptide IHPAHHK. Monoclonalantibody TAB immunoreacts with the TAB epitope located on a portion ofhGPIIb other than the C-terminal hGPIIb fragment, because the materialimmunoreactive with TAB does not become released into the supernatantafter chymotrypsin digestion.

[0177] (2) Isolation of C-Terminal hGPIIb Fragment

[0178] Isolated PMI-1 antibody molecules prepared as described inExample 3c, are covalently linked to Sepharose beads using cyanogenbromide-activated Sepharose (Pharmacia) according to the manufacturer'sinstructions to form PMI-1 Sepharose having about 20 mg of antibodyprotein per ml of packed beads.

[0179] Five ml of chymotrypsin digested platelet suspension, prepared asin Example 1c(1), is admixed with 5 ml of packed PMI-1 Sepharose beadsand the resulting admixture is maintained at 4 C. for 12-18 hours toallow any C-terminal hGPIIb fragment in the suspension to bind to thePMI-1 Sepharose. The PMI-1 Sepharose is then transferred to a columncontaining a scintered glass support, and washed with 25 ml of washbuffer (described in Example 4), also containing 50 mM octylglucoside.Thereafter wash buffer containing 50 mM octylglucoside is added to thecolumn and 1.25 ml fractions are collected as the added buffer comes off(elutes from) the column to form eluted fractions.

[0180] Fifty microliters (ul) of each eluted fraction is then analyzedby sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis(SDS-PAGE) using a 10 - 20% acrylamide gel gradient containing 3% SDSand 5% 2-mercaptoethanol according to the methods of Laemmli, Nature,227:680-685 (1970). Proteins contained in the eluted fractions soanalyzed are transferred from the SDS-PAGE gels to nitrocellulose andvisualized by autoradiography according to the method of Johnson et al,Gen. Anal. Techn., 1:3-8 (1984) using ¹²⁵I-labeled PMI-1. The apparentmolecular weight of the visualized proteins is determined by comparisonto protein molecular weight markers also analyzed on the gel. Allmolecular weights are in units of one thousand daltons.

[0181] Fractions containing eluted proteins that reacted with PMI-1antibody are collected and pooled to form an isolated C-terminal hGPIIbfragment-containing solution, i.e., substantially isolated hGPIIbfragment. The isolated protein exhibited an apparent weight of about3,900 daltons when analyzed by SDS-PAGE, and constituted the majorprotein component in the isolated fragment-containing solution.

[0182] The amino acid residue sequence of the C-terminal hGPIIb fragmentidentified herein after in vitro proteolysis of platelets is shown inSEQ ID NO 1 from residue 173 to residue 200.

[0183] 2. Preparation of Polypeptide Analogs

[0184] Based on the amino acid residue sequence deduced from thenucleotide sequence of hGPIIb, polypeptides corresponding to the varioushGPIIb C-terminal regions utilized herein are chemically synthesized onan Applied Biosystems Model 430A Peptide Synthesizer using thesymmetrical anhydride method of Hagenmaier, et al., Hoppe-Sevler's Z.Phisiol. Chem., 353:1973 (1982).

[0185] The amino acid residue sequences of the various polypeptideanalogs synthesized are shown in Table 1.

[0186] 3. Antibody Production

[0187] a. Preparation of Immunogen

[0188] The term “immunogeno”, as used herein, describes an entity thatinduces antibody production in the host animal. In some instances, theantigen and immunogen are the same entity, while in other instances, thetwo entities are different. Immunogens used to elicit antibodies of thisinvention are whole platelets, platelet membranes, isolated GPIIb-IIIa,isolated C-terminal hGPIIb fragment or hGPIIb polypeptide analogs asdescribed above.

[0189] Polyclonal anti-peptide antibodies are produced by immunizingrabbits with a peptide prepared in Example 2 above and coupled tocarrier protein. Coupling of the peptide with glutaraldehyde tothyroglobulin as antigenic carrier (Bovine Type I, Sigma Chemical Co.,St. Louis, Mo.) is conducted as described by J. G. Dockray, ReaulatoryPeptides, 1:169-186 (1980), which is hereby incorporated by reference toform thyroglobulin coupled peptide.

[0190] b. Preparation of Polyclonal Antibody Compositions

[0191] Immunizations to produce a polyclonal antibody composition aredone according to standard protocols. Briefly, a rabbit is immunizedintradermally at one week intervals with 400 micrograms (ug) ofimmunogen consisting of thyroglobulin coupled peptide at 3 mg/ml. Theimmunogen is diluted 1:1 in Complete Freund's Adjuvant for the firstimmunization, in Incomplete Freund's Adjuvant for the second and thirdimmunizations, and in normal saline for the fourth and fifth.

[0192] Injections are administered with 100 ug distributed over 4 sites.Thereafter, the antibody is harvested and tested according to the assayof Example 4a. In that assay, polyclonal antibody produced using apeptide of Table 1 immunoreacts with the carboxy terminal hGPIIbfragment and with the immunizing peptide, but does not immunoreact withthe polypeptide REQNSLDSWGPK.

[0193] C. Preparation of Monoclonal Antibody Compositions

[0194] Monoclonal antibodies that immunoreact with C-terminal hGPIIbfragment were produced using standard hybridoma technology withexceptions as noted. Balb C mice were immunized biweekly with 50 ug ofplatelet membranes prepared as described by Barber et al., J. Biol.Chem., 245:6357-65 (1970), incorporated herein by reference, emulsifiedalternately in complete and incomplete Freund's adjuvant. Mice were bledfrom the retro-orbital plexus, and sera were screened for antibodies bysolid-phase radioimmunoassay as described in Shadle et al., J. CellBiol., 99:2056-2060 (1984). Three days after the fifth immunization,about 1×10⁸ lymphocytes were isolated from the spleens of mice, admixedinto a suspension and fused with 5×10⁷ P3X63AG8.053 mouse myeloma cellsusing 50% PEG 4000 as the cell fusion promoter. The resultanttransformed (fused) antibody-producing cells (hybridomas) were initiallytransferred to 96-well microtiter plates at a density of about 1×10⁶cells per well and cultured in selective HAT medium.

[0195] Tissue culture supernatant appearing to contain viable HATresistant hybridoma cells after 8 days of culturing were screened in theELISA assay described in Example 4a for the presence of antibodymolecules that immunoreact with GPIIb-IIIa prepared as described inExample 1b. Hybridoma cultures were identified that producedGPIIb-IIIa-immunoreacting antibody molecules. The isolated hybridomaswere then subcloned twice at limiting dilutions to provide about 1 cellper well and 24 of the resultant hybridoma cultures were shown to be ofmonoclonal origin on the basis of three criteria: (1) each supernatantwas from a single cell focus and immunoreacted with GPIIb-IIIa in theELISA screen, (2) each supernatant showed a single homogeneous band whenanalyzed by cellulose acetate gel electrophoresis according to themethod described in Monoclonal Antibodies: Principles and Practice, J.W. Goding, ed., Academic Press, Inc., Orlando, Fla., 1983, and (3) eachsupernatant contained a single isotype of immunoglobulin when analyzedusing the Mouse Ig Screening and Isotyping Kit according to theinstructions provided by the manufacturer, Boehringer-MannheimBiochemicals, Indianapolis, Ind.

[0196] One monoclonal antibody producing hybridoma, PMI-1, isolated bythe above procedure using an inoculum containing platelet membranes isavailable from the ATCC and has an accession number HB 9476.

[0197] A culture of hybridoma cells capable of secreting PMI-1monoclonal antibodies were introduced into a mouse and the ascites fluidwas then harvested as is well known. PMI-1 monoclonal antibody moleculespresent in the harvested ascites were isolated using protein-A Sepharose(Pharmacia, Piscataway, N.J.) according to the manufacturer'sinstructions to form isolated PMI-1 antibody molecules.

[0198] The protein concentration of isolated antibody molecules in aliquid composition was determined, as needed, using the Bio-Rad ProteinAssay Kit (Bio-Rad, Richmond, Calif.) according to the manufacturer'sinstructions.

[0199] To prepare a monoclonal antibody composition containing¹²⁵I-labeled antibody molecules, 350 microliters (ul) of PBS (0.15 MNaCl, 0.01 M sodium phosphate, pH 7.09) containing 1 milligram permilliliter (mg/ml) of the above isolated PMI-1 antibody molecules wereadmixed with 40 micrograms (ug) of chloramine-T and 1 milliCurie (mCi)of carrier-free Na¹²⁵I (Amersham, Arlington Heights, Ill.). Theresulting admixture was maintained for 5 minutes at about 20C. and thenadmixed with 20 ul of a 2 mg/ml sodium metabisulfite solution (2 mg/ml)and 20 ul of a potassium iodide solution. Thereafter, 800 ul of PBScontaining 1% BSA were admixed followed by further admixture ofdiisopropylfluorophosphate to a final concentration of 10 mM. Theresulting admixture was maintained for 60 minutes at 22 C. and thendialyzed against PBS. The specific activity of the resulting¹²⁵I-labeled antibody molecules was about 4.5 microCurie (uCi) per ug.

[0200] Compositions containing Fab fragments were prepared by digestingisolated PMI-1 antibody molecules with papain (200:1 weight per weightof Ig to papain) for 6 hours at 37° C. following the methods of Mage etal., Methods in Enzymology, 70:142-150 (1980). Undigested Ig and Fcfragments were removed by chromatography on protein A-Sepharose. Theresulting Fab fragments-containing compositions were then ready for use,or were ¹²⁵I-labeled, as needed, using the same procedures as describedabove for monoclonal antibody compositions.

[0201] 4. Diagnostic Assays

[0202] a. ELISA To Screen Monoclonal Antibodies

[0203] Antibody molecules contained in hybridoma culture supernatant areexamined for their ability to immunoreact with hGPIIb polypeptideanalogs. Fifty microliters (ul) of coating solution (0.1M NaHCO₃, pH8.0, 0.1% NaN₃) containing 10 ug/ml of polypeptide analogs prepared inExample 2 are admixed into the wells of flat-bottom 96-well microtiterplates (Immulon 2; Dynatech Laboratories, Chantilly, Va.). The platesare then maintained for 60 minutes at 37° C. to permit the GPIIb-IIIa toadsorb onto the walls of the wells. The coating solution is removed byshaking, the wells are rinsed twice with wash buffer (10 mM Tris at pH7.4, 0.05% (v/v) TWEEN-20, 0.15 M NaCl, and 200 mg/ml merthiolate), andthen 200 ul of blocking solution [5% bovine serum albumin (BSA;w/v) incoating solution] are admixed into each well (solid support) to blockexcess protein sites.

[0204] The wells are maintained for 60 minutes at about 37° C. and thenthe blocking solution is removed. About 50 ul of hybridoma culturesupernatant diluted 1:1 in dilution buffer consisting of 0.1% (w/v) BSAin wash buffer is added to each well to form an immunoreactionadmixture. The resulting solid/liquid phase immunoreaction admixturesare maintained at room temperature for 60 minutes to permit formation ofa first solid phase-bound immunoreaction product between the solidphase-bound polypeptide analog and admixed antibodies. The solid andliquid phases are then separated, the wells are rinsed twice with washbuffer, and excess liquid is removed by shaking.

[0205] Fifty ul of a solution containing horseradish peroxidase labeledgoat anti-mouse IgG (Tago Inc., Burlingame, Calif.), diluted 1:1000 indilution buffer is admixed into each well to form a second solid/liquidphase immunoreaction admixture (labeling immunoreaction admixture). Thewells are maintained for 60 minutes at room temperature to permitformation of a second immunoreaction product between the labeledantibody and any solid phase-bound antibody of the first immunoreactionproduct and then rinsed twice with wash buffer to isolate the solidphase-bound label-containing immunoreaction products. Excess liquid isthen removed from the wells.

[0206] Fifty ul of freshly prepared chromogenic substrate solutioncontaining 4.0 mg/ml 0-phenylenediamine and 0.012% (v/v) hydrogenperoxide in CP buffer (243 ml of 0.1 M citric acid and 250 ml of 0.2 Mdibasic sodium phosphate per liter H₂O, pH 5.0) are then admixed intoeach well to form a color developing-reaction admixture. Aftermaintaining the color developing-reaction admixture for 10 minutes atabout 20 C., 50 ul of 2 N H₂SO₄ are admixed into each well to stop thedeveloping-reaction, and the resulting solutions are assayed forabsorbance at 490 nanometers (nm) light wavelength using a Model 310ELISA plate reader (Bio-Tek Instruments, Winooski, Vt.).

[0207] Antibody molecule compositions are considered to contain antibodymolecules immunoreactive with C-terminal hGPIIb fragment if the measuredabsorbance at 490 nm (A490) is at least 6 times above background i.e.,above about 0.3 optical density units when measured at A490.

[0208] b. Competition ELISA Assays to Detect C-Terminal GPIIb HeavyChain Fragment in Body Fluids

[0209] Assays to detect C-terminal hGPIIb fragment can be performed on“platelet-poor” plasma, serum or urine samples without concentration.Urine (serum and plasma are used in similar fashion) samples werecentrifuged at 15,000×g for 5 min at room temp, and the resultingsupernatants were collected to form cell free urine samples. Theresulting cell-free urine samples are substantially free of plateletsand therefore any hGPIIb fragment present in the sample is in the formof soluble, cell-free, hGPIIb fragment. Fifty microliters (ul) ofcoating solution (0.1M NaHCO₃, pH 8.0, 0.1% NaN₃) containing 10 ug/ml ofisolated GPIIb-IIIa prepared as described in Example 1b were admixedinto the wells of flat-bottom 96-well microtiter plates (Immulon 2;Dynatech Laboratories, Chantilly, Va.). The plates were then maintainedfor 60 minutes at 37° C. to permit the GPIIb-IIIa to adsorb onto thewalls of the wells. The coating solution was removed by shaking and 200ul of blocking solution [5% bovine serum albumin (BSA;w/v) in coatingsolution] were admixed into each well (solid support) to block excessprotein sites.

[0210] The wells were maintained for 60 minutes at about 37° C. and thenthe blocking solution was removed by rinsing the wells thrice with washbuffer (10 mM Tris at pH 7.4, 0.05% (v/v) TWEEN-20, 0.15 M NaCl, and 200mg/ml merthiolate). Isolated monoclonal antibody PMI-1 prepared inExample 2c or a control monoclonal antibody (TAB), that immunoreactswith hGPIIb but does not immunoreact with C-terminal hGPIIb fragment,were used in the competition ELISA. TAB was obtained from Dr. R. McEver,University of Texas (San Antonio, Tex.). About 50 ul of monoclonalantibody, diluted 1:1 in incubation buffer consisting of 0.1% (w/v) BSAin wash buffer, was admixed, along with the sample to be tested, to eachwell to form an immunoreaction admixture. The proportions were asfollows:

[0211] (a) 20 ul incubation buffer+10 ul of 20 mM EDTA in incubationbuffer+20 ul of sample prepared in Example 1c(i); and

[0212] (b) 50 ul PMI-1 or TAB monoclonal antibody [1:1000 of 4.5 mg/mlIgG (purified on Staph. A columns)].

[0213] One set of immunoreaction admixtures was prepared using a testsample, and a second set of immunoreaction admixtures was prepared inwhich the sample is replaced with a reference standard comprised of apredetermined amount of either isolated GPIIb-IIIa, if the antibody wasTAB, or polypeptide V43 (PSPSPIHPAHHKRDRRQ), prepared as in Example 2,if the antibody was PMI-1.

[0214] Each immunoreaction admixture was incubated overnight at roomtemperature to permit formation of a first solid phase-boundimmunoreaction product containing the solid phase-bound GPIIb-IIIa andthe admixed antibodies.

[0215] The solid and liquid phases were then separated, the wells wererinsed thrice with washing buffer, and excess liquid was removed byshaking. Fifty ul of a solution containing horseradish peroxidaselabeled goat anti-mouse IgG (Tago Inc., Burlingame, Calif.), diluted1:1000 in incubation buffer was admixed into each well to form a secondsolid/liquid phase immunoreaction admixture (labeling immunoreactionadmixture). The wells were maintained for 60 minutes at room temperatureto permit formation of a second immunoreaction product between thelabeled antibody and any solid phase-bound antibody of the firstimmunoreaction product and then rinsed thrice with wash buffer toisolate the solid phase-bound label-containing immunoreaction products.Excess liquid was then removed from the wells.

[0216] Fifty ul of freshly prepared chromogenic substrate solutioncontaining 4.0 mg/ml 0-phenylenediamine and 0.012% (v/v) hydrogenperoxide in CP buffer (243 ml of 0.1 M citric acid and 250 ml of 0.2 Mdibasic sodium phosphate per liter H₂O, pH 5.0) were then admixed intoeach well to form a color developing-reaction admixture. Aftermaintaining the color developing-reaction admixture for 10 minutes atabout 20 C., 50 ul of 2 N H₂SO₄ were admixed into each well to stop thedeveloping-reaction, and the resulting solutions were assayed forabsorbance at 490 nanometers (nm) light wavelength using a Model 310ELISA plate reader (Bio-Tek Instruments, Winooski, Vt.).

[0217] Absorbance measurements obtained using standard were plottedagainst micromolar (uM) concentration of standard in the immunoreactionadmixture to produce a standard curve. Absorbance measurements obtainedusing samples were compared to the standard curve to produce a valueexpressed as equivalents of standard.

[0218]FIG. 1 shows results of the above competition ELISA using samplescontaining C-terminal hGPIIb fragment that were formed as described inExample 1c(1). The data is expressed as equivalents of standard (inmicromoles) detectable in the sample during the time course of thechymotrypsin digestion. Using polypeptide V43 as a standard incombination with antibody PMI-1, antigenic material that corresponds toC-terminal hGPIIb fragment was released into the supernatant during thecourse of the digest (FIG. 1A). In contrast, no detectable antigens werereleased in the supernatant that immunoreact with the TAB antibody (FIG1B).

[0219] Urine samples from a normal donor in the form of cell free urinesamples as described above were tested in the above competition ELISAusing various dilutions of urine in incubation buffer from undiluted to1 part urine to 7 parts buffer (1:8). Equivalents of peptide V43detectable in the urine using PMI-1 were 1.5 uM at 1:8, 3.8 uM at 1:4and 1.5 uM at 1:2. From the data, a normalized value of C-terminalhGPIIb fragment in undiluted urine was 14.4 uM for the normal donor'surine. Thus levels of C-terminal hGPIIb fragment in urine that aresignificantly greater than about 15 uM, such as a 10% increase overnormal values, is indicative of thromotic activity above normal levels.

[0220] Anti-peptide antibodies prepared as described in Example 3c usingthe polypeptides shown in Table 1 are used in the competition ELISA inplace of monoclonal antibody PMI-1, and using the antibody'scorresponding polypeptide rather than isolated GPIIb-IIIa as thesolid-phrase antigen. In this assay, samples containing competingantigen, i.e., C-terminal hGPIIb fragment, or containing standardcomprised of the same corresponding peptide, are measured as above forthe presence and amount of C-terminal hGPIIb fragment. By thiscompetition ELISA assay, anti-peptide antibodies that immunoreact with apolypeptide shown in Table 2 are shown to also immunoreact with isolatedGPIIb/IIIa and with C-terminal hGPIIb fragment prepared in Example 1c(2)

[0221] Although the present invention has now been described in terms ofcertain preferred embodiments, and exemplified with respect thereto, oneskilled in the art will readily appreciate that various modifications,changes, omissions and substitutions may be made without departing fromthe spirit thereof. It is intended, therefore, that the presentinvention be limited solely by the scope of the following claims.

Sequence Listing

[0222] SEQ ID NO 1 illustrates the nucleotide and corresponding aminoacid residue sequence of a cDNA that codes for a portion of GPIIb heavychain (hGPIIb) wherein the nucleotide sequence is shown from left toright and in the direction of 5′ terminus to 3′ terminus using thesingle letter nucleotide base code represented as an uninterruptedlinear series of bases from base 1 to base 600. The amino acid residuesequence is shown from left to right and in the direction fromamino-terminus to carboxy-terminus using the single letter amino acidresidue code represented as an uninterrupted linear series of residuesfrom residue 1 (G) at the amino-terminus to residue 200 (K) at thecarboxy-terminus.

[0223] The reading frame is indicated by the placement of the deducedamino acid residue sequence below the nucleotide sequence such that thesingle letter that represents each amino acid residue is located belowthe first base in the corresponding codon.

[0224] The nucleotide base sequence shown from base 1 through base 600in SEQ ID NO 1 corresponds to the nucleotide sequence shown in FIG. 2 ofPoncz et al., J. Biol. Chem., 262:8476-8482 (1987), from base 2060 tobase 2659. The amino acid residue sequence from residue 1 to residue 200shown in SEQ ID NO 1 corresponds to the sequence shown in FIG. 2 ofPoncz et al., supra, from residue 656 to residue 855. SEQ ID NO 1:GGCGCCCACTACATGCGGGCCCTAAGCAATGTCGAGGGCTTTGAGAGACTCATCTGTAAT 60G  A  H  Y  M  R  A  L  S  N  V  E  G  F  E  R  L  I  C  N 20CAGAAGAAGGAGAATGAGACCAGGGTGGTGCTGTGTGAGCTGGGCAACCCCATGAAGAAG 120Q  K  K  E  N  E  T  R  V  V  L  C  E  L  G  N  P  M  K  K 40AACGCCCAGATAGGAATCGCGATGTTGGTGAGCGTGGGGAATCTGGAAGAGGCTGGGGAG 180N  A  Q  I  G  I  A  M  L  V  S  V  G  N  L  E  E  A  G  E 60TCTGTGTCCTTCCAGCTGCAGATACGGAGCAAGAACAGCCAGAATCCAAACAGCAAGATT 240S  V  S  F  Q  L  Q  I  R  S  K  N  S  Q  N  P  N  S  K  I 80GTGCTGCTGGACGTGCCGGTCCGGGCAGAGGCCCAAGTGGAGCTGCGAGGGAACTCCTTT 300V  L  L  D  V  P  V  R  A  E  A  Q  V  E  L  R  G  N  S  F 100CCAGCCTCCCTGGTGGTGGCAGCAGAAGAAGGTGAGAGGGAGCAGAACAGCTTGGACAGC 360P  A  S  L  V  V  A  A  E  E  G  E  R  E  Q  N  S  L  D  S 120TGGGGACCCAAAGTGGAGCACACCTATGAGCTCCACAACACTGGCCCTGGGACTGTGAAT 420W  G  P  K  V  E  H  T  Y  E  L  H  N  T  G  P  G  T  V  N 140GGTCTTCACCTCAGCATCCACCTTCCGGGACAGTCCCAGCCCTCCGACCTGCTCTACATC 480C  L  H  L  S  I  H  L  P  G  Q  S  Q  P  S  D  L  L  Y  I 160CTGGATATACAGCCCCAGGGGGCGCTTCAGTGCTTCCCACAGCCTCCTGTCAATCCTCTC 540L  D  I  Q  P  Q  G  A  L  Q  C  F  P  Q  P  P  V  N  P  L 180AAGGTGGACTGGGGGCTGCCCATCCCCAGCCCCTCCCCCATTCACCCGGCCCATCACAAG 600K  V  D  W  G  L  P  I  P  S  P  S  P  I  H  P  A  H  H  K 200

[0225]

1 3 600 base pairs nucleic acid single linear cDNA NO NO CDS 1..600/standard_name= “Portion of GPIIb heavy chain” 1 GGC GCC CAC TAC ATG CGGGCC CTA AGC AAT GTC GAG GGC TTT GAG AGA 48 Gly Ala His Tyr Met Arg AlaLeu Ser Asn Val Glu Gly Phe Glu Arg 1 5 10 15 CTC ATC TGT AAT CAG AAGAAG GAG AAT GAG ACC AGG GTG GTG CTG TGT 96 Leu Ile Cys Asn Gln Lys LysGlu Asn Glu Thr Arg Val Val Leu Cys 20 25 30 GAG CTG GGC AAC CCC ATG AAGAAG AAC GCC CAG ATA GGA ATC GCG ATG 144 Glu Leu Gly Asn Pro Met Lys LysAsn Ala Gln Ile Gly Ile Ala Met 35 40 45 TTG GTG AGC GTG GGG AAT CTG GAAGAG GCT GGG GAG TCT GTG TCC TTC 192 Leu Val Ser Val Gly Asn Leu Glu GluAla Gly Glu Ser Val Ser Phe 50 55 60 CAG CTG CAG ATA CGG AGC AAG AAC AGCCAG AAT CCA AAC AGC AAG ATT 240 Gln Leu Gln Ile Arg Ser Lys Asn Ser GlnAsn Pro Asn Ser Lys Ile 65 70 75 80 GTG CTG CTG GAC GTG CCG GTC CGG GCAGAG GCC CAA GTG GAG CTG CGA 288 Val Leu Leu Asp Val Pro Val Arg Ala GluAla Gln Val Glu Leu Arg 85 90 95 GGG AAC TCC TTT CCA GCC TCC CTG GTG GTGGCA GCA GAA GAA GGT GAG 336 Gly Asn Ser Phe Pro Ala Ser Leu Val Val AlaAla Glu Glu Gly Glu 100 105 110 AGG GAG CAG AAC AGC TTG GAC AGC TGG GGACCC AAA GTG GAG CAC ACC 384 Arg Glu Gln Asn Ser Leu Asp Ser Trp Gly ProLys Val Glu His Thr 115 120 125 TAT GAG CTC CAC AAC ACT GGC CCT GGG ACTGTG AAT GGT CTT CAC CTC 432 Tyr Glu Leu His Asn Thr Gly Pro Gly Thr ValAsn Gly Leu His Leu 130 135 140 AGC ATC CAC CTT CCG GGA CAG TCC CAG CCCTCC GAC CTG CTC TAC ATC 480 Ser Ile His Leu Pro Gly Gln Ser Gln Pro SerAsp Leu Leu Tyr Ile 145 150 155 160 CTG GAT ATA CAG CCC CAG GGG GCG CTTCAG TGC TTC CCA CAG CCT CCT 528 Leu Asp Ile Gln Pro Gln Gly Ala Leu GlnCys Phe Pro Gln Pro Pro 165 170 175 GTC AAT CCT CTC AAG GTG GAC TGG GGGCTG CCC ATC CCC AGC CCC TCC 576 Val Asn Pro Leu Lys Val Asp Trp Gly LeuPro Ile Pro Ser Pro Ser 180 185 190 CCC ATT CAC CCG GCC CAT CAC AAG 600Pro Ile His Pro Ala His His Lys 195 200 17 amino acids amino acidunknown peptide internal 2 Pro Ser Pro Ser Pro Ile His Pro Ala His HisLys Arg Asp Arg Arg 1 5 10 15 Gln 200 amino acids amino acid linearprotein 3 Gly Ala His Tyr Met Arg Ala Leu Ser Asn Val Glu Gly Phe GluArg 1 5 10 15 Leu Ile Cys Asn Gln Lys Lys Glu Asn Glu Thr Arg Val ValLeu Cys 20 25 30 Glu Leu Gly Asn Pro Met Lys Lys Asn Ala Gln Ile Gly IleAla Met 35 40 45 Leu Val Ser Val Gly Asn Leu Glu Glu Ala Gly Glu Ser ValSer Phe 50 55 60 Gln Leu Gln Ile Arg Ser Lys Asn Ser Gln Asn Pro Asn SerLys Ile 65 70 75 80 Val Leu Leu Asp Val Pro Val Arg Ala Glu Ala Gln ValGlu Leu Arg 85 90 95 Gly Asn Ser Phe Pro Ala Ser Leu Val Val Ala Ala GluGlu Gly Glu 100 105 110 Arg Glu Gln Asn Ser Leu Asp Ser Trp Gly Pro LysVal Glu His Thr 115 120 125 Tyr Glu Leu His Asn Thr Gly Pro Gly Thr ValAsn Gly Leu His Leu 130 135 140 Ser Ile His Leu Pro Gly Gln Ser Gln ProSer Asp Leu Leu Tyr Ile 145 150 155 160 Leu Asp Ile Gln Pro Gln Gly AlaLeu Gln Cys Phe Pro Gln Pro Pro 165 170 175 Val Asn Pro Leu Lys Val AspTrp Gly Leu Pro Ile Pro Ser Pro Ser 180 185 190 Pro Ile His Pro Ala HisHis Lys 195 200

What is claimed is:
 1. A substantially isolated C-terminal hGPIIbfragment that has a molecular weight of about 3,900 daltons, comprisingan amino acid residue sequence that corresponds to the carboxy terminalportion of the amino acid residue sequence of hGPIIb shown in SEQ ID NO1, including the amino acid residue sequence represented by the formula-IHPAHHK-, said fragment having the capacity to immunoreact with themonoclonal antibody, PMI-1.
 2. The hGPIIb fragment of claim 1 whereinsaid amino acid residue sequence comprises the amino acid residuesequence shown in SEQ ID No 1 from about residue 173 to about residue200.
 3. A hGPIIb analog comprising a polypeptide of no more than about200 residues, said polypeptide having an amino acid residue sequencethat corresponds to the sequence of hGPIIb shown in SEQ ID NO 1 andincludes at least about 7 contiguous amino acid residues from theamino-acid residue sequence of hGPIIb shown in SEQ ID NO 1 from residue173 to residue
 200. 4. The analog of claim 3 wherein said polypeptidecomprises an amino acid residue sequence corresponding to the formula:PQPPVNPLK,       PLKVDWGLPIP,               PIPSPSPIHPAHHK, or                     IHPAHHK,

having the amino acid residue sequence shown in SEQ ID NO 1 fromresidues 173 to 181, from residues 179 to 189, from residues 187 to 200,or from residues 194 to 200, respectively.
 5. The analog of claim 3wherein said polypeptide has an amino acid residue sequence thatcorresponds to the amino acid residue sequence shown in SEQ ID NO 1 fromabout residue 1 to about residue
 200. 6. The analog of claim 3 whereinsaid polypeptide has an amino acid residue sequence represented by theformula: PQPPVNPLK,       PLKVDWGLPIP,               PIPSPSPIHPAHHK, or                     IHPAHHK,

having the amino acid residue sequence shown in SEQ ID NO 1 fromresidues 173 to 181, from residues 179 to 189, from residues 187 to 200,or from residues 194 to 200, respectively.
 7. An antibody comprisingantibody molecules that immunoreact with a polypeptide having an aminoacid residue sequence as shown in SEQ ID NO 1 from residue 173 toresidue 193, and do not substantially immunoreact with a polypeptiderepresented by the formula REQNSLDSWGPK having the amino acid residuesequence shown in SEQ ID NO 1 from residue 113 to residue
 124. 8. Theantibody of claim 7 wherein said antibody is a monoclonal antibody.
 9. Amethod for detecting the presence of C-terminal hGPIIb fragment in abody fluid sample containing cells, comprising the steps of: a)separating the cells from said body fluid sample to form a cell-freebody fluid sample; b) admixing an aliquot of said cell-free body fluidsample with a composition that contains an antibody that: i)immunoreacts with said C-terminal hGPIIb fragment that includes an aminoacid residue sequence as shown in SEQ ID NO 1 from residue 173 toresidue 200, and ii) does not substantially immunoreact with apolypeptide represented by the formula REQNSLDSWGPK having the aminoacid residue sequence shown in SEQ ID NO 1 from residue 113 to residue124, to form an immunoreaction admixture; c) maintaining saidimmunoreaction admixture under biological assay conditions for a timeperiod sufficient to form an immunoreaction product; and d) detectingthe presence of said immunoreaction product, and thereby the presence ofsaid fragment.
 10. The method of claim 9, wherein said body fluid isvascular fluid and said cells include platelets.
 11. The method of claim9, wherein said body fluid is urine.
 12. The method of claim 9, whereinsaid antibody comprises antibody molecules immunoreactive with apolypeptide having an amino acid sequence that corresponds to one of thefollowing formula: PQPPVNPLK,             PLKVDWGLPIP,               PIPSPSPIHPAHHK, or                       IHPAHHK,

having the amino acid residue sequence shown in SEQ ID NO 1 fromresidues 173 to 181, from residues 179 to 189, from residues 187 to 200,or from residues 194 to 200, respectively.
 13. The method of claim 9,wherein said antibody composition comprises a monoclonal antibody,PMI-1, produced by a hybridoma having ATCC accession number HB
 9476. 14.The method of claim 9 wherein said admixing in step (b) includesadmixing said body fluid sample and said composition with a solidsupport comprising a solid matrix having affixed thereto a polypeptidehaving an amino acid residue sequence that includes (1) a C-terminalhGPIIb fragment according to claim 1 or (2) a hGPIIb analog according toclaim 3 such that said immunoreaction admixture is a competitionimmunoreaction admixture having a liquid phase and a solid phase, andsaid immunoreaction product formed in step (c) is in the solid phase.15. The method of claim 14 wherein said antibody is a labeled antibody,having a label affixed to the antibody.
 16. The method of claim 15wherein said detecting in step (d) comprises determining the presence ofsaid label in the solid phase immunoreaction product, and thereby thepresence of said immunoreaction product.
 17. The method of claim 14wherein said polypeptide has an amino acid residue sequence shown in SEQID NO 1 from residue 173 to residue
 200. 18. The method of claim 14wherein said polypeptide is the platelet glycoprotein GPIIb-IIIa. 19.The method of claim 9 wherein said detecting in step (d) comprises thesteps of: (i) admixing said immunoreaction product formed in step (c)with an indicating means to form a second reaction admixture; (ii)maintaining said second reaction admixture for a time period sufficientfor said indicating means to bind to the immunoreaction product formedin step (c) and form a second reaction product; and (iii) determiningthe presence of said indicating means in said second reaction product,and thereby the presence of said immunoreaction product formed in step(c).
 20. The method of claim 19 wherein said indicating means is alabeled antibody comprising an antibody having a label affixed thereto.21. A diagnostic system in kit form for assaying for the presence ofC-terminal hGPIIb fragment in a body fluid sample, comprising a packagecontaining, in an amount sufficient to perform at least one assay, anantibody composition comprising antibody molecules that: a) immunoreactwith a polypeptide having an amino acid residue sequence as shown in SEQID NO 1 from residue 173 to residue 193, and b) do not substantiallyimmunoreact with a polypeptide represented by the formula REQNSLDSWGPKhaving the amino acid residue sequence shown in SEQ ID NO 1 from residue113 to residue
 124. 22. The diagnostic system of claim 21 wherein saidantibody is affixed to a solid matrix.
 23. The diagnostic system ofclaim 21 that further includes a solid support comprised of a solidmatrix having affixed thereto a polypeptide having an amino acid residuesequence that includes (1) a C-terminal hGPIIb fragment according toclaim 1 or (2) a hGPIIb analog according to claim 3, said polypeptidecapable of immunoreacting with said antibody molecules.
 24. Thediagnostic system of claim 23 that further includes, in a separatepackage, labeled specific binding agent for signaling the presence of animmunoreaction product in the solid phase.
 25. A diagnostic system inkit form for assaying for the presence of C-terminal hGPIIb fragment ina body fluid sample, comprising separate packages containing, in anamount sufficient to perform at least one assay, (i) an antibodycomposition comprising antibody molecules that: a) immunoreact with apolypeptide having an amino acid residue sequence as shown in SEQ. ID NO1 from residue 173 to residue 193, and b) do not substantiallyimmunoreact with a polypeptide represented by the formula REQNSLDSWGPKhaving the amino acid residue sequence shown in SEQ ID NO 1 from residue113 to residue 124; and (ii) a polypeptide having an amino acid residuesequence that includes (1) a C-terminal hGPIIb fragment according toclaim 1 or (2) a hGPIIb analog according to claim 3, said polypeptidecapable of immunoreacting with said antibody molecules.
 26. Thediagnostic system of claim 25 that further includes a solid supportcomprised of a solid matrix having said polypeptide affixed thereto.